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HEMATOPOIESIS
From the Division of Cellular and Molecular Biology,
Ontario Cancer Institute; Department of Laboratory Medicine and
Pathobiology, Toronto General Hospital; Departments of Medical
Biophysics and Laboratory Medicine and Pathobiology, University of
Toronto; Toronto, ON; Cancer Biology Program and Division of
Hematology-Oncology, Department of Medicine, Beth Israel Deaconess
Medical Center; and Departments of Pediatric Oncology and Adult
Oncology, Dana-Farber Cancer Institute; Boston, MA.
Erythropoietin (EPO) specifically activates the Janus kinase JAK2
and the transcription factor signal transducer and activator of
transcription-5 (STAT5). All members of the STAT family are tyrosine
phosphorylated in response to cytokine stimulation at a conserved
carboxy-terminal tyrosine, Y694, in the case of STAT5. To determine
structural features important for STAT signaling, we generated an
activation-specific STAT5 antibody using a phosphopeptide containing
amino acids 687 to 698 of STAT5 as antigen. This antibody specifically recognizes tyrosine- phosphorylated STAT5 but not nonphosphorylated STAT5. In immunoprecipitation reactions from cell
lines and primary erythroblasts, 2 distinct polyclonal
activation-specific STAT5 antibodies selectively immunoprecipitate the
tyrosine phosphorylated EPO receptor (EPO-R) in addition to STAT5 under
native and denaturing conditions. We propose that the
activation-specific STAT5 antibody recognizes the 2 substrates to which
the STAT5 SH2 domain interacts, namely, the tyrosine- phosphorylated
EPO-R and STAT5 itself. Several studies have implicated EPO-R Y343,
Y401, Y431, and Y479 in the recruitment of STAT5. Using a series of
EPO-R tyrosine mutants expressed in Ba/F3 cells, we have shown that the
activation-specific STAT5 antibody immunoprecipitates an EPO-R
containing only 2 tyrosines at positions 343 and 401, confirming the
importance of these tyrosines in STAT5 recruitment. These data uncover
a novel aspect of STAT SH2 domain recognition and demonstrate the
utility of activation-specific antibodies for examining the specificity
of STAT-cytokine receptor interactions.
(Blood. 2001;97:2230-2237) Erythropoietin (EPO), the primary cytokine
regulator of erythropoiesis, exerts its biological function by binding
to its cognate receptor, a 66-kd single transmembrane
receptor.1 Despite undergoing ligand-dependent tyrosine
phosphorylation, the EPO receptor (EPO-R) and other members of the
cytokine receptor family do not contain a tyrosine kinase catalytic
domain within their cytoplasmic regions. The identification of the
Janus family of tyrosine kinases has revealed a mechanism by which
hematopoietic cytokines activate intracellular tyrosine
phosphorylation. Several studies have shown that EPO specifically
activates Janus kinase-2 (JAK2).2,3 The critical
importance of EPO,4 EPO-R,4,5 and
JAK2 6,7 in erythropoiesis have been demonstrated through
gene targeting strategies; deletion of any of these genes gives rise to
embryonic lethality because of an inability of the mice to successfully undergo the transition from primitive to definitive erythropoiesis.
Stimulation of JAK2 catalytic activity results in the tyrosine
phosphorylation of several tyrosine residues of the EPO-R cytoplasmic tail.8 After the EPO-R is tyrosine phosphorylated, SH2
domain-containing proteins such as signal transducer and activator of
transcription-5 (STAT5),9-15 Ship1,16
Shp1,17 Shp2,18,19 and the 85-kd subunit of
phosphatidylinositol 3' kinase20-22 are recruited to specific sites.
Elegant studies, first performed in the interferon signaling system,
proposed the following mechanism of STAT activation.23 In
resting cells, STAT proteins are cytosolic and are not normally phosphorylated. Cytokine-dependent JAK activation results in tyrosine phosphorylation of several cytoplasmic tyrosine residues of the cytokine receptor, some of which may represent consensus binding sites
for the SH2 domain of particular STAT proteins. Specific STAT proteins
are recruited to the cytokine receptor in an SH2-dependent fashion.
When in the proximity of the active JAK kinase, the STAT protein
becomes tyrosine phosphorylated at a conserved position. Through an
unknown mechanism, the STAT protein disassociates from the
cytokine receptor, undergoes reciprocal SH2-mediated homodimerization, and shuttles rapidly to the nucleus.
Several studies have shown that EPO-dependent JAK2 activation
results in tyrosine phosphorylation of STAT5.9-15 Using a
variety of techniques including analysis of EPO-R deletion mutants,
EPO-R tyrosine mutants, and phosphopeptide competitions in
electrophoretic mobility shift assays, the interaction sites of STAT5
on the EPO-R were proposed to include Y343, Y401, Y431, and
Y479.9,12-14 However, more careful mutagenesis studies
have suggested that Y343 and Y401 are the major STAT5 binding
sites.13,14 EPO-R Y343 is sufficient for STAT5 tyrosine
phosphorylation.9
Phosphopeptide analysis demonstrated that STAT1 becomes tyrosine
phosphorylated at a unique position, Y701.24 Indeed, all STAT proteins contain a conserved tyrosine located carboxy-terminal to
the SH2 domain, which becomes tyrosine phosphorylated upon cytokine
stimulation and participates in SH2 domain-mediated homodimerization or heterodimerization (Y694 for STAT5A, Y699 for
STAT5B).25 To examine the importance of this motif for
STAT5 signaling, we synthesized a tyrosine-phosphorylated peptide and
generated an antibody that specifically recognizes
tyrosine-phosphorylated but not unactivated STAT5. Herein, we show that
this antibody recognizes tyrosine-phosphorylated STAT5 after
interleukin-3 (IL-3) or EPO stimulation in Western blotting analysis.
The activation-specific STAT5 antibody also faithfully
immunoprecipitates tyrosine-phosphorylated STAT5. Significantly, the
activation-specific STAT5 antibody also immunoprecipitates the
tyrosine-phosphorylated EPO-R. We hypothesized that this antibody may
selectively recognize the 2 substrates of the STAT5 SH2 domain, namely,
the tyrosine-phosphorylated EPO-R and STAT5 itself. The ability of the
activation-specific STAT5 antibody to immunoprecipitate several EPO-R
tyrosine mutants was examined. These experiments revealed that the
activation-specific antibody was capable of immunoprecipitating an
EPO-R containing only Y343 and Y401.
Reagents
Cells and cell culture
Various EPO-R constructs were electroporated into Ba/F3 cells. Individual G418-resistant subclones were isolated by limiting dilution. The expression of EPO-R was confirmed by Western blotting using a peptide-specific EPO-R antibody (Santa Cruz, Santa Cruz, CA), and the EPO-dependent growth characteristics of each subclone were examined by performing an XTT assay as described.26 Generation of phenylhydrazine-primed splenic erythroblasts C57Bl/6 mice were injected intraperitoneally on days 1 and 2 with a sterile solution of phenylhydrazine hydrochloride (6 mg/mL) in alpha-minimal essential medium to achieve a dose of 60 mg/kg.27 The mice were killed on day 5 by cervical dislocation. The spleen was removed, and a single-cell suspension was generated. The cells were incubated in alpha-minimal essential medium containing 2% fetal calf serum and 50 µM -mercaptoethanol for 4 hours. Cells were stimulated with 50 U/mL EPO for various periods, and
lysates were generated as described previously.3
Generation of EPO-R mutants EPO-R tyrosine mutants were generated via overlap extension polymerase chain reaction. These included a series of single tyrosine mutants in which phenylalanine was substituted for different tyrosine residues in the EPO-R and a series of add-back mutants to an EPO-R devoid of tyrosine residues. Oligonucleotide primers were selected that produced a phenylalanine at amino acid positions 343, 401, 431, or 479. Polymerase chain reaction was performed using pBluescript SK-EPO-R or selected pBluescript SK-EPO-R tyrosine mutants to generate to SphI-EcoRI fragment in pCR-Script. The fidelity of all constructs was confirmed by sequencing both strands of the 440-base pair fragment. Each SphI-EcoRI fragment was subcloned into SphI-EcoRI-digested pBluescript SK-EPO-R. The full-length EPO-R cDNA was then subcloned into pCDNA3 using KpnI and EcoRI.Antibody generation A peptide was synthesized that contained amino acids 687 to 698 of ovine STAT5. A cysteine residue was added to the N-terminus of the peptide and was used to conjugate the peptide to bovine serum albumin using a succinimide ester. Rabbits were immunized with the peptide-bovine serum albumin conjugate. A second activation-specific STAT5 antibody (71-6900) was generously provided by Dr Sarah Guadagno.Immunoprecipitation and Western blotting The indicated cell lines were incubated in the absence of cytokine for 4 hours and then stimulated with the corresponding cytokine: 10 ng/mL murine IL-3, 50 U/mL human EPO, 5 ng/mL murine interferon- . Cell lysis, immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blotting was carried out as described.3
Immunoblots were performed with the activation-specific STAT5 antibody
at 1:5000 dilution followed by horseradish peroxidase (HRP)-conjugated
protein A (1:5000). For detection of tyrosine phosphorylation, the
monoclonal antiphosphotyrosine 4G10 antibody was used (1 µg/mL)
followed by HRP-sheep antimouse immunoglobulin G (1:5000). For
detection of STAT5 protein, antibody to STAT5 (1:5000) was incubated
followed by an incubation with the appropriate HRP-protein A. Immunoreactive proteins were stripped from nitrocellulose membranes by
incubation in 62.5 mM Tris-HCl (pH 6.8), 2% (wt/vol) SDS, and 100 mM
Immunoprecipitations were performed on 2 × 107 cell equivalents of each protein lysate. Cells were lysed in a buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 10 mM Na4P2O7, 10 mM NaF, 1 mM Na3VO4, 5 mM ethylenediaminetetraacetic acid (EDTA), 2 µg/mL aprotinin, 2 µg/mL leupeptin, 2 µg/mL pepstatin A, and 1 mM phenylmethylsulfonyl fluoride (PMSF) (buffer A) for preparation of nondenaturing lysates. The samples were incubated on ice for 5 minutes and then centrifuged at 13 000g for 5 minutes. For generation of denatured extracts, 2 × 107 cells were boiled for 5 minutes in 250 µL of a buffer containing 50 mM Tris-HCl (pH 8.0), 0.5% SDS, 10 mM Na4P2O7, 10 mM NaF, 1 mM Na3VO4, 5 mM EDTA, 2 µg/mL aprotinin, 2 µg/mL leupeptin, 2 µg/mL pepstatin A, and 1 mM PMSF. Then, 750 µL of 50 mM TrisHCl (pH 8.0), 150 mM NaCl, 0.5% sodium deoxycholate, 1.0% Triton X-100 containing the above phosphatase, and protease inhibitors were added. The samples were rotated for 5 minutes and then centrifuged at 14 000g for 15 minutes. The activation-specific STAT5 antibody was added to either lysate, and an immunoprecipitation was performed. Samples were resolved by SDS-PAGE, and the identity of tyrosine-phosphorylated proteins and STAT proteins were analyzed by immunoblotting as described above. Electrophoretic mobility shift assays Nuclear extracts were prepared from Ba/F3-EPO-R subclones stimulated in the presence or absence of 10 ng/mL IL-3 or 50 U/mL EPO. Cell pellets were resuspended in 1.0 mL buffer B (10 mM HEPES [pH 7.9], 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 1 mM PMSF, 5 µg/mL aprotinin, 5 µg/mL leupeptin, and 5 µg/mL pepstatin A) and allowed to swell on ice for 10 minutes. Cells were then vortexed for 10 seconds, centrifuged at 10 000g for 10 seconds, and the supernatant was discarded. The pellet was washed once in buffer B, and then nuclear proteins were resuspended in an appropriate volume of buffer C (20 mM HEPES [pH 7.9], 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 1 mM PMSF, 5 µg/mL aprotinin, 5 µg/mL leupeptin, and 5 µg/mL pepstatin A).Gel shift experiments were performed as described28 with a
double-stranded 32P-labeled oligonucleotide derived from
the
Activation-specific STAT5 antibody identifies tyrosine- phosphorylated STAT5 in a variety of hematopoietic cell lines To characterize the specificity of the activation-specific STAT5 antibody, Western blotting was performed on lysates from several cytokine-dependent hematopoietic cell lines. Cells were depleted of cytokine for 4 hours and then stimulated for 10 minutes and analyzed by Western blotting (Figure 1). IL-3 and EPO stimulation of either Ba/F3-EPO-R (lanes 2 and 3) or DA-3-EPO-R (lanes 6 and 7) cells and EPO stimulation of HCD-57 cells (lane 11) resulted in the phosphorylation of a 95-kd phosphoprotein detected by the activation-specific STAT5 antibody. All 3 cell lines respond to IFN- and IFN- (data not shown); however, at this exposure STAT5 phosphorylation in response to IFN- could only be detected in HCD-57
cells (lane 12). Long exposures revealed a weak IFN--stimulated tyrosine phosphorylation of STAT5 (data not shown). The
activation-specific STAT5 antibody also weakly recognized a 140-kd
phosphoprotein after IL-3 stimulation, likely the
tyrosine-phosphorylated IL-3 receptor (lanes 2 and 6) and a truncated
form of STAT5 expressed in DA-3-EPO-R cells (lanes 5 and 6). Truncated
versions of STAT5 have been previously been described in myeloid cell
lines including DA-3,30 32D,31
FDC-P1,32,33 and WEHI-3 33,34 as well as
primary monocytes.35 No other cytokine-stimulated
phosphoproteins were detected using the activation-specific STAT5
antibody. The identity of the 95-kd phosphoprotein was confirmed by
stripping and reprobing the blot with a peptide-specific STAT5 antibody (lower panel).
The kinetics of activation of STAT5 was analyzed in Ba/F3-EPO-R
cells (Figure 2). IL-3 and EPO led to the
rapid induction of a 95-kd phosphoprotein as detected by immunoblot
analysis with the activation-specific STAT5 antibody. The
EPO-dependent tyrosine phosphorylation of STAT5 was maximal at 15 minutes (lane 10).
Ba/F3-EPO-R cells were incubated with increasing concentrations of
either murine IL-3 or human EPO to examine the concentration dependence
of STAT5 tyrosine phosphorylation (Figure
3). Concentrations as low as 200 pg/mL
IL-3 (lane 2) or 0.5 U/mL EPO (lane 9) resulted in tyrosine
phosphorylation of STAT5 consistent with the biologically active
concentrations of these cytokines.
A common epitope is shared by STAT5 and the EPO-R in EPO-stimulated cells To determine whether proteins other than STAT5 contain a similar phosphotyrosine motif after EPO stimulation, an immunoprecipitation experiment was performed (Figure 4A). Lysates were prepared under native and denaturing conditions, and immunoprecipitations were performed with 2 distinct activation-specific STAT5 polyclonal antibodies to exclude the possibility of unique reactivity of a single antiserum (see "Materials and methods"). Precipitating proteins were initially analyzed by antiphosphotyrosine Western blotting. Immunoprecipitations performed with 2 distinct activation-specific STAT5 antibodies revealed that each antibody immunoprecipitated STAT5 (lanes 4 and 6). However, an additional 72-kd phosphoprotein, the EPO-R, was immunoprecipitated by both activation-specific STAT5 antibodies (lanes 4 and 6) from nondenatured lysates. To determine whether the activation-specific STAT5 antibodies could directly immunoprecipitate the EPO-R, experiments were also performed from denatured lysates. The EPO-R was directly immunoprecipitated from these lysates using both activation-specific antibodies (lanes 10 and 12). Importantly, a peptide-specific antibody that recognized total STAT5 did not immunoprecipitate the EPO-R from either lysate preparation (lanes 2 and 8). No other cytokine-inducible phosphoproteins were detected in this experiment.
The ability of the activation-specific STAT5 antibody to immunoprecipitate STAT and the EPO-R from primary erythroblasts was examined (Figure 4B). Phenylhydrazine causes an anemia when administered in vivo, which results in an elevation in splenic EPO-responsive erythroblasts.27 As illustrated above, the activation-specific STAT5 antibody selectively immunoprecipitates EPO-R and STAT5 after EPO stimulation of the primary cells (lanes 2 and 4). These experiments indicate that 2 distinct activation-specific STAT5 antibodies are capable of immunoprecipitating both EPO-R and STAT5 after EPO stimulation. This suggested that either the EPO-R was being coprecipitated in a complex with STAT5 or that an epitope on EPO-R is shared with the tyrosine phosphorylation site of STAT5. Coprecipitation of STAT5 with the EPO-R has been demonstrated; however, the fraction of tyrosine-phosphorylated EPO-R shown to associate with STAT5 is relatively small, in keeping with the transient nature of STAT association with cytokine receptors.36,37 Given that the activation-specific STAT5 antibodies selectively recognized tyrosine phosphorylated STAT5 and EPO-R but no other EPO-dependent substrates, we became intrigued with the possibility that this antibody selectively recognizes the 2 substrates to which the STAT5 SH2 domain binds, STAT5 and EPO-R. Alternatively, a small fraction of EPO-R and STAT5 could associate, which would account for these observations. Data from studies using various EPO-R deletion and tyrosine substitutions suggest that there is considerable redundancy in the ability of EPO-R to recruit and activate STAT5. As many as 4 distinct tyrosines have been implicated in STAT5 binding, including EPO-R Y343 (Y1), Y401 (Y2), Y431 (Y4), and Y479 (Y8). Some of this evidence was garnered by the addition of specific phosphopeptides to electrophoretic mobility shift assays. More careful mutagenesis approaches revealed that EPO-dependent tyrosine phosphorylation of Y343 and Y401 accounts for most STAT5 binding.13,14 To examine whether the activation-specific STAT5 antibody could
immunoprecipitate the tyrosine-phosphorylated EPO-R in the absence of
transfected STAT5, reconstitution experiments were performed in 293T
cells (Figure 5). For this experiment, we
used wild-type EPO-R, EPO-R containing STAT5 binding sites (EPO-R
Y1Y2Y4Y8), a complementary mutant containing tyrosine to phenylalanine
substitutions (EPO-R F1F2F4F8), and a full-length EPO-R construct
devoid of cytoplasmic tyrosine residues (EPO-R F8). Transient
transfections were performed with the various EPO-R constructs in the
presence or absence of JAK2 and/or STAT5. The cells were depleted of
serum 24 hours after transfection and then stimulated in the presence or absence of EPO. Lysates were prepared, and immunoprecipitations were
performed with the activation-specific STAT5 antibody. EPO-dependent signaling was established in this transient assay because EPO could
stimulate the tyrosine phosphorylation of EPO-R and STAT5 (lane 2). The
activation-specific STAT5 antibody immunoprecipitated both STAT5 and
the wild-type EPO-R after EPO stimulation (lane 2). Importantly, the
activation-specific antibody selectively immunoprecipitated the EPO-R
in the absence of exogenous STAT5 (lane 4). EPO-R Y1Y2Y4Y8 (lane 6) was
immunoprecipitated by the antibody, whereas EPO-R F1F2F4F8 (lane 8) and
EPO-R F8 (lane 10) were not recognized by the activation-specific STAT5
antibody after EPO stimulation. JAK2 was required for EPO-R tyrosine
phosphorylation (lane 12), and no tyrosine-phosphorylated proteins were
observed in mock transfected cells (lanes 13 and 14). This experiment
clearly showed that the activation-specific STAT5 antibody could
directly immunoprecipitate the EPO-R in the absence of transfected
STAT5. Similar results were obtained for the Zymed activation-specific STAT5 antibody (data not shown).
We were interested in identification of the epitope on EPO-R that is
shared with the tyrosine phosphorylation site of STAT5. Having mapped
the association site in the previous experiment to EPO-R Y1, Y2, Y4, or
Y8, we selected 4 distinct mutants: EPO-R Y1Y2Y4Y8, EPO-R Y1Y2Y4, EPO-R
Y1Y2, and EPO-R F8 (Figure 6A).
We examined the tyrosine phosphorylation of the EPO-R in the various cell lines by performing an immunoprecipitation with a peptide-specific EPO-R antibody followed by antiphosphotyrosine immunoblotting (Figure 6B). All of the EPO-R tyrosine mutants, with the exception of EPO-R F8, were tyrosine phosphorylated after EPO stimulation. The level of phosphorylation of EPO-R Y1Y2Y4 was slightly lower (lane 8), which probably reflects lower expression of the EPO-R in this particular subclone. To examine the expression of the EPO-R in the selected subclones, an EPO-R Western blot was performed using lysates from each cell line (Figure 6C). Comparable expression of the EPO-R was observed in each lane, with slightly lower expression of EPO-R Y1Y2Y4 (lanes 5 and 6). XTT assays revealed that all EPO-R mutants with the exception of EPO-R F8 displayed EPO-dependent proliferation (data not shown). We next examined whether these mutations disrupted the epitope shared with tyrosine-phosphorylated STAT5. Each cell line was stimulated in the presence or absence of EPO, lysates were made, and immunoprecipitations were performed with the activation-specific STAT5 antibody (Figure 6D). STAT5 was tyrosine phosphorylated in response to EPO in cells expressing all of the EPO-R constructs with the exception of EPO-R F8. The identity of STAT5 was confirmed by stripping and reprobing the blot with a peptide-specific STAT5 antibody. Furthermore, the 72-kd EPO-R was observed in all of the activation-specific STAT5 immunoprecipitations with the exception of EPO-R F8. This confirms that tyrosine phosphorylation of the EPO-R at Y1 or Y2 is sufficient for STAT5 activation, as previously reported.13,14 The activation-specific STAT5 antibody is very selective because it can immunoprecipitate an EPO-R containing tyrosines at Y1 (Y343) and Y2 (Y401), which represent the optimal STAT5 recruitment sites.13,14 To confirm that EPO-dependent STAT5 tyrosine phosphorylation correlated
with DNA binding, an electrophoretic mobility shift assay was performed
(Figure 7). Nuclear extracts were
prepared from each cell line stimulated in the absence of cytokine or
in the presence of IL-3 or EPO. A complex was observed after
stimulation of Ba/F3-EPO-R cells with either IL-3 (lane 2) or EPO
(lane 3). This oligonucleotide-DNA complex could be competed with an
excess of unlabeled oligonucleotide (lane 4) but not a nonspecific
oligonucleotide (lane 5). The presence of STAT5 in the complex was
confirmed by the addition of a peptide-specific STAT5 antibody, which
resulted in supershifting of the band to a higher mobility.
Ba/F3-EPO-R Y1Y2Y4Y8, Ba/F3-EPO-R Y1Y2Y4, and Ba/F3-EPO-R Y1Y2 cells
all stimulated a STAT5-specific complex after EPO stimulation. There
was a slight increase in EPO-dependent STAT5 activation in Ba/F3-EPO-R
F8, similar to previous reports.14
Several studies have shown that EPO activates tyrosine phosphorylation and DNA binding of STAT5 in a time- and dose-dependent manner.9-15 Transformation by Friend spleen focus-forming virus has also been shown to correlate with constitutive activation of STAT5.38 STAT proteins are cytosolic and unphosphorylated under resting conditions.39 EPO-dependent JAK2 activation initiates a cascade of tyrosine phosphorylation that results in phosphorylation of the EPO-R and several critical intracellular substrates. Phosphorylation of specific motifs on the EPO-R results in the recruitment of STAT5 to the EPO-R and subsequent tyrosine phosphorylation by JAK2. Tyrosine phosphorylation allows STAT5 to disassociate from the EPO-R and undergo a reciprocal SH2-mediated dimerization. The STAT5 dimer is shuttled to the nucleus via an unknown mechanism and has been shown to transcriptionally regulate oncostatin M,40 Cis,41 and cyclin D1.42 The binding specificity of several SH2 proteins was first described by an elegant affinity chromatography approach. Using this method it was shown that SH2 domains bound to distinct phosphotyrosine sequences and the specificity was conferred by amino acids located immediately on the carboxyl-terminal side of the phosphotyrosine.43,44 Unfortunately, the cytosolic transcription factors, STATs, are refractory to this analysis, so the identification of STAT SH2 binding sites has evolved through the mutagenesis of tyrosine sequences found in various cytokine receptors. In this study, we have shown that polyclonal antibodies raised against
a phosphorylated peptide corresponding to the STAT5 tyrosine
phosphorylation site recognize the tyrosine-phosphorylated STAT5
but not the unphosphorylated STAT5. In addition, 2 distinct polyclonal
antibody preparations displayed the unexpected finding of also
recognizing the tyrosine-phosphorylated EPO-R in cell lines and primary
splenic-derived erythroblasts in immunoprecipitation experiments. The
activation-specific STAT5 antibodies did not recognize any other
EPO-stimulated phosphoproteins in Western blotting experiments. Thus,
the activation-specific STAT5 antibodies appear to recapitulate the
selectivity of the STAT5 SH2 binding sites, namely, the EPO-R and STAT5
itself. Neither antibody appears to function as a nonspecific
antiphosphotyrosine antibody because other EPO-dependent
tyrosine-phosphorylated substrates such as phospholipase
C Previous studies have shown that EPO activates the tyrosine
phosphorylation of STAT5 in transfected cell lines including
Ba/F3-EPO-R,9,14 32D-EPO-R,53
DA-3-EPO-R,9,12,53 and FDCP-EPO-R,13
and in erythroid cell lines including HCD-57,36,54
UT-7,11,55,56 and human colony-forming
units-erythroid.57 Phenylhydrazine administration
generates a population of splenic erythroblasts that accurately
represents erythroid differentiation as monitored by the expression of
EPO-R, GATA-1, EKLF, NF-E2, Several cytokines, including EPO,9-15
thrombopoietin,59,60 IL-3,53,61
IL-5,61 granulocyte-macrophage colony-stimulating factor,55,61 IL-2,62,63 growth
hormone,55,64-66 and prolactin,55 all confer
activation of STAT5. The ability of such a wide range of cytokines to
induce the tyrosine phosphorylation of STAT5 relies on the expression
and tyrosine phosphorylation of the appropriate binding motifs in all
of these receptors. In addition to the aforementioned studies completed
on EPO, mutagenesis experiments of the IL-2R The crystal structures of STAT1 71 and
STAT3 The significant finding of this study was the ability of 2 unique activation-specific STAT5 antibodies to selectively recognize the tyrosine-phosphorylated EPO-R and STAT5 in immunoprecipitation experiments. Despite a lack of obvious sequence similarity between the STAT5 SH2 substrates found on the EPO-R and STAT5, there is strong recognition of both proteins by the antibody after EPO stimulation. Perhaps tyrosine phosphorylation of STAT5 exerts a conformational change within the SH2 domain of STAT5. This may account for why the unphosphorylated STAT5 molecule would have a distinct binding specificity as compared with when STAT5 is tyrosine phosphorylated. This hypothesis must await the solution of monomeric, unactivated, and dimeric tyrosine-phosphorylated STAT5 crystal structures.
We thank Eleanor Fish for the gift of murine IFN-
Submitted June 8, 2000; accepted November 27, 2000.
Supported by grants from the Medical Research Council of Canada (D.L.B.), University of Toronto Connaught New Staff Grant (D.L.B.), National Institutes of Health DK 50693 (B.G.N.), F32 DK 09465 (M.Y.), and CA 79547 (D.A.F.). D.A.F. received funding from the Brent Leahey Fund.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Dwayne L. Barber, Ontario Cancer Institute, Division of Cellular and Molecular Biology, 610 University Ave, Toronto, ON, M5G 2M9, Canada; e-mail: dbarber{at}oci.utoronto.ca.
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Abstract
Introduction
-aminolevulinic acid synthase,
signals emanating from the erythropoietin receptor.
Eur J Biochem.
1997;249:637-647